Redox-enabled electronic interrogation and feedback control of hierarchical and networked biological systems

Microelectronic devices can directly communicate with biology, as electronic information can be transmitted via redox reactions within biological systems. By engineering biology’s native redox networks, we enable electronic interrogation and control of biological systems at several hierarchical levels: proteins, cells, and cell consortia. First, electro-biofabrication facilitates on-device biological component assembly. Then, electrode-actuated redox data transmission and redox-linked synthetic biology allows programming of enzyme activity and closed-loop electrogenetic control of cellular function. Specifically, horseradish peroxidase is assembled onto interdigitated electrodes where electrode-generated hydrogen peroxide controls its activity. E. coli’s stress response regulon, oxyRS, is rewired to enable algorithm-based feedback control of gene expression, including an eCRISPR module that switches cell-cell quorum sensing communication from one autoinducer to another—creating an electronically controlled ‘bilingual’ cell. Then, these disparate redox-guided devices are wirelessly connected, enabling real-time communication and user-based control. We suggest these methodologies will help us to better understand and develop sophisticated control for biology.


Notes -Addendums for Figure 2 in Main Manuscript
1.In Fig. 2f, we examined the difference in current obtained during electroinduction (-0.8 V) with or without exogenous oxygen supplement.Surface-assembled E. coli (NB101 + pOxy-sfGFP, OD600 = 6), submerged in 20% LB, was acclimatized in the custom environmental chamber at 34°C for 1.5 hours prior to electroinduction.The blue curve shows the output current without exogenous oxygen supplement (~0.5 μA, indicating minimal H2O2 generation), while the purple curve shows the current during which we supplied 0.4 ft 3 /h (~0.01 m 3 /h) of oxygen (starting from 0 s) through built-in tubing in the connector of the 4-well optoelectrochemical device.In the case of supplemented oxygen, it took about 2 minutes, but the measured current was observed to increase steadily.This profile is likely due to the active metabolism of the E. coli cells entrapped in the artificial biofilm that depleted the dissolved oxygen in the culture media.After two minutes, increased current shows that oxygen was present at the electrode surface, suggesting sufficient oxygen for respiratory function.Considering that the current for the controls without cells (both gel and no-gel) was always higher than the gel with cells, this indicates that the cells did indeed metabolize provided oxygen.Thus, we designed the 3D-printed optoelectrochemical device to include built-in tubing for exogenous oxygen supply.Schematic illustrations of the entry ports are shown below (in Supplementary Fig. 3).We believe that both oxygen diffusion and the gentle stirring (convection) caused by the airflow facilitated oxygen transport, and together, sufficient oxygen could be delivered to the electrode surface allowing both cell respiration and electroinduction.
2. In Fig. 2e, we chose -0.8 V as the applied voltage to generate hydrogen peroxide (H2O2) in the ITO-based electrochemical platform.As shown above (in Supplementary Fig. 2), applying -0.8 V to 150 μL of 20% LB in our custom device produced the highest level of H2O2.Additionally, we observed a visible color change to the electrode (from transparent to yellowish-brown) after applying -1 V to the ITO electrodes.The observed color change is also reported in previous studies and will likely affect the resistivity or other properties

Notes
1.The fluorescence response (on a per cell level; we divided the total fluorescence from each homogenous culture by its OD600) of eCRISPRa GFP cells (NB101 harboring pSC-O108, pdCas9ω, and pMC-GFP) induced with various concentrations of peroxide as indicated in Supplementary Fig. 4a (i).We observed an increase in fluorescence intensity with peroxide and saw the response plateaued at higher peroxide concentrations (50-200 μM).
2. OD600 values of the samples noted above are also provided in Supplementary Fig. 4a (ii).
Although we found significant statistical differences in OD600 between the uninduced (0 μM) and induced (6.25-200 μM) cultures at hour 4, these differences are small (e.g., median

Notes -Addendums for Figure 3 in Main Manuscript
1.In addition to Fig. 3d, we included the raw integrated density (sum of the values from each pixel) obtained from the FITC-channel confocal images in Fig. 3c as a representative of the total fluorescence recorded in each image (Supplementary Fig. 6b).We observed similar trends to the increase in ON% with increasing charge that is shown in Fig. 3d.
2. The green fluorescence intensity of a single "cell" (or, perhaps more accurately, single "cell-cluster") is shown in Supplementary Fig. 6c by analyzing the mean gray value from six randomly-chosen cells in Fig. 3.The area (pixel size) of the cells is depicted alongside in Supplementary Fig. 6d.We found the size of "ON" cells is statistically uniform, and their fluorescence intensity exhibited a similar trend to the data depicting raw integrated density (Supplementary Fig. 6b).
3. The number of "ON" cells and total cells are shown in Supplementary Fig. 6e and 6f.We observed similar trends between 6a (ON%; "ON"/total cells), 6b (total fluorescence in field of view), and 6e (number of "ON" cells).Compared to the uninduced sample, we found the total cell count of the samples receiving 30-minute induction and 200 μM peroxide induction were slightly but significantly lower than the uninduced, suggesting again that peroxide exhibited low cytotoxicity.This observation also agrees with the OD600 data in Supplementary Fig. 4a (ii).
4. In Fig. 3d-f, we reported the total applied charge for each individual experiment.We believe the charge variations from the experiments results from slight differences in electrode surface area and material, potential deviation from the reference electrodes, and contents of the culture media.used one-way ANOVA, assuming the replicates were normally distributed, and P values were calculated between induced samples and the uninduced control.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.Exact P values can be found in the source data.

Notes
1. Growth curves of the 'bilingual' cells are shown in Supplementary Fig. 7d (iii).We observed attenuated final cell growth (at 7 h) as well as a relative decrease in growth rate for the cultures with more peroxide addition.Specifically, at hour 7, the uninduced samples (OD600 ~ 2.32) had a slightly higher OD600 than the samples with three (OD600 ~ 2.16) or four (OD600 ~ 2.12) inductions.

Notes -Addendums for Figure 4 in Main Manuscript
In Fig. 4d, we reported the total applied charge for the electroinduced samples.For the 0 h and 0&3 h samples, we applied -0.8 V for 30 minutes (resulting in a charge of 2.71 mC) to both cultures immediately after deposition.After 3 hours of incubation at 37°C, we administered an additional 30 minutes of -0.8 V electroinduction to the 0&3 h sample (resulting in an additional charge of 0.43 mC, making a total of 3.14 mC).We believe the diminished charge observed for the second application reflects the decrease of available oxygen at the electrode surface, because the cells were actively respiring and no oxygen was supplemented to the samples in this experiment.

Notes -Addendums for Figure 5 in the Main Manuscript
In Fig. 5e, we showed that the information "written" by WE1 (i.e., generated peroxide) can be "stored" in our electro-biochemical device and "retrieved" by WE2 at a later time.For this, we performed data writing (on WE1) and recording (on WE2) for a prolonged period (Supplementary Fig. 8).First, -0.8 V was applied to WE1 for 600 s to generate peroxide.0 V was then applied to WE2 for peroxide detection, and we observed minimal decrease in WE2 current throughout the entire recording period (from 600 s to 3600 s for the #1 round).Next, this process (both data writing and recording) was repeated immediately (denoted the #2 round) and we saw an increase in WE2 current (throughout the entire 3600 s recording period), indicating that the increased peroxide level (from the #2 round of data writing) was stored and detected.It also computes the ratio between the current slope and the maximum slope (Smax).If two consecutive ratios fall below the user-set limit (expressed as a ratio), the algorithm considers the threshold met and initiates electro-induction via the potentiostat.The Smax will, in turn, return to 0 and a new cycle will begin.

Supplementary
immediately after the upcoming fluorescence measurement (for a user-defined duration or charge).The elapsed time for this occurrence was typically 15 minutes.Otherwise, the remote algorithm sends a SMS verification message to alert the human users and seek permission to terminate the experiment.
User: Human users can respond to a query by sending text messages on a mobile phone (by replying "Y "or "N") to indicate whether the experiment should be terminated or not.If replied with "Y", BioSpark initiates the photobleaching program as a demonstration to destroy the expression product.

Supplementary Figure 2 .
H2O2 production under different applied voltage.Voltage was applied for 5 minutes to 150 μL of 20% LB.Data are presented as mean±s.d.(n = 4).

of the electrode 1 .Supplementary Figure 3 .
Schematic of the mechanism for exogenous oxygen supply.(a) Side view of an individual sample well in the optoelectrochemical device.(b) Top view of an individual sample well in the optoelectrochemical device.Supplementary Figure 4. H2O2-inducible CRISPR activation of gfpmut2 (a) Fluorescence (i) and OD600 (ii) of NB101 harboring pSC-O108, pdCas9ω, and pMC-GFP measured at 2 hours (green) and 4 hours (purple) after H2O2 induction.Data are presented as mean±s.d.(n = 5).Filled triangle (2 h) and circles (4 h) represent individual replicates.Solid lines represent the fitted curves.EC50 values represent the half-maximal effective concentration of n = 5 H2O2 inductions of CRISPRa.Data in (ii) were analyzed by ordinary two-way ANOVA.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.Exact P values can be found in the source data.(b) Relative fold change of gRNA (sg108) expression induced with various levels of H2O2.Data are presented as mean±s.d.(n = 3).Open circles represent individual replicates.
OD600 of the uninduced and 200 μM sample are 1.04 and 0.95, respectively) and concluded peroxide (from 0-200 μM) exhibited minimal cytotoxicity to the eCRISPRa cells under current experimental conditions.Supplementary Figure 5. H2O2-inducible CRISPR activation of lasI (a) AI-1 produced by NB101 harboring pSC-O108, pdCas9ω, and pMC-lasI-LAA after being induced by 0 μM (blue) and 200 μM (yellow) of H2O2.Data are presented as mean±s.d.(n = 3).Open circles represent individual replicates.(b) AI-1 bioassay standard curve.Data are presented as mean±s.d.(n = 3).Calibration curve was generated via linear interpolation of experimental data, and the dashed lines represent the 95% confidence interval.(c) Normalized fluorescence response from AI-1 inducible strain (NEB10β + LasR_S129T-GFPmut3).Data are presented as mean±s.d.(n = 3).The fitted curve was generated via linear interpolation of experimental data, and the dashed lines represent the 95% confidence interval.Supplementary Figure 6.Analysis of eCRISPRa confocal images depicted in Fig. 3c of the main manuscript.(a) Percentage of E. coli in the PEG-SH film that were activated through eCRISPRa.Data are adapted from Fig. 3d.(b) Raw integrated density obtained from FITCchannel images in Fig. 3c.(c) Mean gray value of individual "ON" cells (n = 6).(d) Pixel size of each "ON" cell assessed in (c).Data are presented as box plots (center line at the median, upper bound at 75 th percentile, lower bound at 25 th percentile) with whiskers at minimum and maximum values.Each open circle represents one assessed "ON" cell.(e) Number of "ON" cells.(f) Number of total cells.Comparisons used ordinary one-way ANOVA.Comparisons between all samples are shown except for (f), where only the comparisons between the uninduced (0) and the induced were included.Data are presented as mean±s.d.(n = 4; n = 6 for (c) and (d)).Individual replicates were indicated as open circles.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant.Exact P values can be found in the source data.

Figure 8 .
Voltages applied on WE1 and WE2 of the electro-biochemical device (from Fig. 5e).Dark blue: applied voltage on WE1.Pink: applied voltage on WE2.Blue shaded area: round 1 (#1) of data writing and recording.Green shaded area: round 2 (#2) of data writing and recording.(arrows).Fluorescence values from 0 -1 h was normalized to the fluorescence of the uninduced control at time 0. Fluorescence values from 1 -4 h was normalized to each sample's fluorescence at the beginning of each cycle (i.e., 1 or 3 h).Data are presented as mean±s.d.(n ≥ 3).Individual replicates were indicated by the open circles.(b) Growth curve of all experimental samples from repetitive inductions.Peroxide addition is indicated below its corresponding time.Filled circles (indicating one dose of peroxide), triangles (two doses), and squares (three doses) represent the mean.Error bars represent the standard deviation (n = 3).fluorescence measurements.The algorithm stores and updates the value of the maximum slope.